Project Summary We propose to systematically investigate and characterize sequence elements that are necessary for genome function in their native context in contrast to existing high- throughput assays of genome function that detect sequence elements that are sufficient for function. We will accomplish this goal with three specific aims. We will develop a novel Multiplexed Editing Regulatory Assay (MERA) to test the effect of thousands of targeted mutations in native regulatory regions in a single experiment (Aim 1). We will use MERA to characterize the regulation of key developmental genes, the function of selected regulatory elements, and the gene expression effects of SNPs that are discovered in GWAS studies (Aim 2). Using these data we will build a model that will allow us to predict the bases that comprise the necessary genome and estimate the effect of non-coding genotype on gene expression (Aim 3). Through our new experimental and computational method we will help lay the groundwork for a novel paradigm in revealing the effect of human variation at base pair resolution.